Human Medullary Thyroid Carcinoma Cell Research Articles

Abstract 4665: USP18 promotes anlotinib resistance in medullary thyroid carcinoma by stabilizing aurora B kinase

Abstract Targeted therapy resistance is the main cause of disease progression in advanced medullary thyroid carcinoma (MTC). However, the key molecules and mechanisms remain unclear. We previously performed a genome-wide in vitro screen of Anlotinib resistance-related genes in human MTC cell line TT using CRISPR-dCas9-SAM system and identified ubiquitin-specific protease 18 (USP18) as a key molecule mediating Anlotinib resistance in MTC. However, its downstream mechanism needs further clarification. Based on the high-throughput DNA sequencing results of the control group and the Anlotinib screening group, a group of key genes related to Anlotinib resistance in MTC was screened through the MAGeCK algorithm and the enrichment ratio of sgRNA in the Anlotinib screening group. Among them, USP18 ranked first in the enrichment results. Analysis of the correlation between the expression level of USP18 in MTC patient specimens and survival revealed that patients with high USP18 expression in tumor cells had poorer prognosis. Overexpression of USP18 in MTC tumor cells significantly promoted the growth of subcutaneous xenograft tumors in NOD/SCID mice treated with Anlotinib. In vitro cell experiments also showed that overexpression of USP18 promoted the proliferation of MTC tumor cells and significantly inhibited the proportion of cell apoptosis after Anlotinib treatment. Further research found that USP18, as a deubiquitinating enzyme that can specifically remove ISG15-like ubiquitin-like proteins from substrate proteins, can improve the kinase stability of Aurora B by removing ISG15-like ubiquitin modification and promote MTC tumor cell proliferation through the activation of PI3K-Akt signaling regulated by Aurora B kinase. Application of Aurora B kinase inhibitor in Anlotinib-resistant MTC cell lines significantly inhibited tumor cell proliferation, and Aurora B kinase inhibitor significantly increased the proportion of cell apoptosis in MTC tumor cells overexpressing USP18. In the subcutaneous xenograft tumor model of MTC in NOD/SCID mice, the growth of xenograft tumors in the Anlotinib combined with Aurora B kinase inhibitor treatment group was also significantly inhibited. In summary, overexpressed USP18 can activate PI3K-Akt signaling and promote tumor cell proliferation by increasing the stability of Aurora B kinase, ultimately leading to the formation of Anlotinib resistance in MTC patients. Combined use of Aurora B kinase inhibitor is a potentially effective combined treatment strategy for Anlotinib-resistant MTC patients. Citation Format: Xuan Qin, Xing Wan, Dapeng Li, Yimeng Liu, Xianhui Ruan, Fangkun Luan, Kejia Xu, Jia Li, Rong Xiang, Yongjun Piao, Yi Shi, Xiangqian Zheng. USP18 promotes anlotinib resistance in medullary thyroid carcinoma by stabilizing aurora B kinase [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4665.

Preclinical Evaluation of Novel Tyrosine-Kinase Inhibitors in Medullary Thyroid Cancer.

Simple SummaryMedullary thyroid carcinoma (MTC) is a neuroendocrine tumor arising from parafollicular calcitonin-secreting C cells of the thyroid. Most of the patients affected by MTC, especially the familial form, harbor a mutation of the RET proto-oncogene. In patients with advanced disease, medical therapy is represented by two tyrosine-kinase inhibitors: cabozantinib and vandetanib. However, their usage is limited by several adverse events and drug-resistance onset. The aim of this preclinical study was to evaluate the antitumor activity of novel molecules for the therapy of MTC: SU5402, an inhibitor of the fibroblast growth factor receptor type 1 (FGFR-1) and vascular endothelial growth factor receptor (VEGFR)-2; sulfatinib, a multi-target kinase inhibitor selective for FGFR-1 and the VEGFR-1, -2, and -3; SPP86, a RET-specific inhibitor. Our results suggest a potential role in targeting the FGFR and VEGFR signaling pathways as an alternative strategy for resistant tumors and a significative antitumor activity of this new RET-specific inhibitor.Medullary thyroid carcinoma (MTC) is a neuroendocrine tumor arising from parafollicular C cells of the thyroid gland. In this preclinical study, we tested three tyrosine-kinase inhibitors (TKIs): SU5402, a selective inhibitor of fibroblast growth factor receptor (FGFR)-1 and vascular endothelial growth factor receptor (VEGFR)-2; sulfatinib, an inhibitor of FGFR-1 and VEGFR-1, -2, -3; and SPP86, a RET-specific inhibitor. The effects of these compounds were evaluated in vitro in two human MTC cell lines (TT and MZ-CRC-1), and in vivo using xenografts of MTC cells in zebrafish embryos. SU5402, sulfatinib and SPP86 decreased cell viability. Sulfatinib and SPP86 significantly induced apoptosis in both cell lines. Sulfatinib and SPP86 inhibited the migration of TT and MZCRC-1 cells, while SU5402 was able to inhibit migration only in TT cells. In vivo we observed a significant reduction in TT cell-induced angiogenesis in zebrafish embryos after incubation with sulfatinib and SPP86. In conclusion, sulfatinib and SPP86 displayed a relevant antitumor activity both in vitro and in vivo. Moreover, this work suggests the potential utility of targeting FGFR and VEGFR signaling pathways as an alternative therapy for MTC.

Analogs of the Heat Shock Protein 70 Inhibitor MKT-077 Suppress Medullary Thyroid Carcinoma Cells.

Medullary thyroid carcinoma (MTC) is a neuroendocrine tumor mainly caused by mutations in the RET proto-oncogene. We previously demonstrated that depletion of the mitochondrial molecular chaperone, mortalin, can effectively suppress human MTC cells in culture and in mouse xenografts, by disrupting mitochondrial bioenergetics and subsequently inducing apoptosis and RET downregulation. Similar effects were induced by MKT-077, a water-soluble rhodocyanine dye analog known to inhibit mortalin, but with notable toxicity in animals. These observations led us to evaluate recently developed MKT-077 analogs that exhibited higher selectivity to HSP70 proteins and improved bioavailability. We validated the MTC cell-suppressive effects of mortalin depletion in three-dimensional cultures of the human MTC lines, TT, and MZ-CRC-1, and then evaluated different MKT-077 analogs in two- and three-dimensional cell cultures, to show that the MKT-077 analogs, JG-98 and JG-194, effectively and consistently inhibited propagation of TT and MZ-CRC-1 cells in these cultures. Of note, these compounds also effectively suppressed the viability of TT and MZ-CRC-1 progenies resistant to vandetanib and cabozantinib. Moreover, JG-231, an analog with improved microsomal stability, consistently suppressed TT and MZ-CRC-1 xenografts in mice. These data suggest that mortalin inhibition may have therapeutic potential for MTC.

Salinomycin and its derivatives as potent RET transcriptional inhibitors for the treatment of medullary thyroid carcinoma (MTC).

Rearranged during transfection kinase (RET) is a validated molecular target in medullary thyroid cancer (MTC), as activating mutations in RET are often associated with the development of MTC. The present study reports the first preclinical characterization of salinomycin and selected analogs as potent RET transcriptional inhibitors. Reverse transcription‐PCR and immunoblotting revealed that salinomycin profoundly decreased RET expression in the TT human MTC cell line by inhibiting RET transcription. Moreover, salinomycin resulted in remarkable anti‐proliferative activity against MTC that is driven by RET (gain of function mutation) by selectively inhibiting the intracellular PI3K/Akt/mTOR signaling pathway. Also, flow cytometry and fluorescence‐activated cell sorting showed that salinomycin induces G1 phase arrest and apoptosis by reducing the expression of retinoblastoma protein, E2F1, cyclin D and CDK4. The structure‐activity relationship of salinomycin was investigated in this study. Some of the salinomycin derivatives showed the ability to reduce RET expression where others fail to alter RET expression. These results suggest that the RET‐suppressing effect of salinomycin may be largely attributed to disruption of the Wnt pathway, presumably through interference with the ternary LRP6‐Frizzled‐Wnt complex. Furthermore, these findings support the further preclinical evaluation of salinomycin and its analogs as a promising new class of therapeutic agents for the improved treatment of MTCSupport or Funding InformationAmerican Cancer Society for their support of this study (grant no. RSGM‐12‐046‐01‐CDD).

Cofilin is a mediator of RET-promoted medullary thyroid carcinoma cell migration, invasion and proliferation

Medullary thyroid carcinoma (MTC) is a rare neuroendocrine tumor that originates from parafollicular thyroid C cells and accounts for 5% of thyroid cancers. In inherited cases of MTC, and in about 40% of sporadic cases, activating mutations of the receptor tyrosine kinase proto-oncogene RET are found. Constitutively active RET triggers signaling pathways involved in cell proliferation, survival and motility, but the mechanisms underlying malignant transformation of C-cells have been only partially elucidated.Cofilin is a key regulator of actin cytoskeleton dynamics. A crucial role of cofilin in tumor development, progression, invasion and metastasis has been demonstrated in different human cancers, but no data are available in MTC. Interestingly, RET activation upregulates cofilin gene expression.The aim of this study was to investigate cofilin contribution in invasiveness and growth of MTC cells, and its relevance in the context of mutant RET signaling.We found that cofilin transfection in human MTC cell line TT significantly increased migration (178 ± 44%, p < 0.001), invasion (165 ± 28%, p < 0.01) and proliferation (146 ± 18%, p < 0.001), accompanied by an increase of ERK1/2 phosphorylation (2.23-fold) and cyclin D1 levels (1.43-fold). Accordingly, all these responses were significantly reduced after genetic silencing of cofilin (−55 ± 10% migration, p < 0.001, −41 ± 8% invasion, p < 0.001, −17 ± 3% proliferation, p < 0.001). These results have been confirmed in primary cells cultures obtained from human MTCs.The inhibition of constitutively active RET in TT cells by both the RET pharmacological inhibitor RPI-1 and the transfection of dominant negative RET mutant (RETΔTK) resulted in a reduction of cofilin expression (−37 ± 8%, p < 0.001 and −31 ± 16%, p < 0.01, respectively). Furthermore, RPI-1 inhibitory effects on TT cell migration (−57 ± 13%, p < 0.01), but not on cell proliferation, were completely abolished in cells transfected with cofilin.In conclusion, these data indicate that an unbalanced cofilin expression, induced by oncogenic RET, contributes to promote MTC invasiveness and growth, suggesting the possibility of targeting cofilin pathway for more effective treatment of MTC.

Cofilin is a mediator of RET-promoted medullary thyroid carcinoma cell migration, invasion and proliferation

Abstract Medullary thyroid carcinoma (MTC) is a rare neuroendocrine tumor that originates from parafollicular thyroid C cells and accounts for 5% of thyroid cancers. In inherited cases of MTC, and in about 40% of sporadic cases, activating mutations of the receptor tyrosine kinase proto-oncogene RET are found. Constitutively active RET triggers signaling pathways involved in cell proliferation, survival and motility, but the mechanisms underlying malignant transformation of C-cells have been only partially elucidated. Cofilin is a key regulator of actin cytoskeleton dynamics. A crucial role of cofilin in tumor development, progression, invasion and metastasis has been demonstrated in different human cancers, but no data are available in MTC. Interestingly, RET activation upregulates cofilin gene expression. The aim of this study was to investigate cofilin contribution in invasiveness and growth of MTC cells, and its relevance in the context of mutant RET signaling. We found that cofilin transfection in human MTC cell line TT significantly increased migration (178 ± 44%, p The inhibition of constitutively active RET in TT cells by both the RET pharmacological inhibitor RPI-1 and the transfection of dominant negative RET mutant (RETΔTK) resulted in a reduction of cofilin expression (−37 ± 8%, p In conclusion, these data indicate that an unbalanced cofilin expression, induced by oncogenic RET, contributes to promote MTC invasiveness and growth, suggesting the possibility of targeting cofilin pathway for more effective treatment of MTC.

SAT-566 Cofilin Is a Mediator of RET-Promoted Medullary Thyroid Carcinoma Cell Migration, Invasion and Proliferation

Medullary thyroid carcinoma (MTC) is a rare neuroendocrine tumor that originates from parafollicular thyroid C cells, accounting for 5% -10% of thyroid cancers. In all inherited cases of MTC, and in about 40% of sporadic cases, activating mutations of the receptor tyrosine kinase proto-oncogene RET are found. Constitutively active RET triggers signaling pathways involved in cell motility, proliferation and survival, but the mechanisms underlying malignant transformation of C-cells have been only partially elucidated. Cofilin is a key regulator of actin cytoskeleton dynamics. A crucial role of cofilin in tumor development, progression, invasion and metastasis has been demonstrated in different human cancers, but no data are available in MTC. Interestingly, RET activation upregulates cofilin gene expression. The aim of this study was to investigate cofilin contribution in invasiveness and growth of MTC cells, and its relevance in the context of mutant RET signaling. We found that cofilin transfection in human MTC cell line TT significantly increased migration (178.5±44%, p<0.001), invasion (194±27%, p<0.05) and proliferation (145.9±18%, p<0.001), accompanied by an increase of ERK1/2 phosphorylation (1.8-fold) and cyclin D1 levels (1.43-fold). Accordingly, all these responses were significantly reduced after genetic silencing of cofilin (-55±10% migration, p<0.001, -40.7±8% invasion, p<0.001, -17±2.6% proliferation, p<0.001). The inhibition of constitutively active RET in TT cells by both the RET pharmacological inhibitor RPI-1 and the transfection of dominant negative RET mutant (RETΔTK) resulted in a reduction of cofilin expression (-30.8±11%, p<0.01 and -31±16%, p<0.01, respectively). Furthermore, RPI-1 inhibitory effects on TT cell migration (-70±18%, p<0.001) and proliferation (-29.4±9% , p<0.01) were completely abolished in cells transfected with cofilin. In conclusion, these data indicate that an unbalanced cofilin expression, induced by oncogenic RET, contributes to promote MTC invasiveness and growth, suggesting the possibility of targeting cofilin pathway for more effective treatment of MTC.

Abstract 4783: Selective antitumor activity of NSC311152 toward medullary thyroid cancer by downregulating the transcription of RET oncogene

Abstract The RET (REarranged during Transfection) proto-oncogene encodes a tyrosine-kinase receptor specifically expressed in tissues originated from neuroectodermal tissues. Dominant-activating mutations in this RET proto-oncogene is often associated with Multiple Endocrine Neoplasia type 2A and 2B and Familial Medullary Thyroid Carcinoma syndromes, characterized by the occurrence of medullary thyroid carcinomas. Human medullary thyroid carcinoma cell line TT cell line harbours a MEN2A-type mutation, so this cell line could be a useful cell system to investigate the antitumor activity of new agents interfering with the function of RET oncogene in medually thyroid cancer. Recently, ellipticine and its derivatives were identified as excellent RET G-quadruplex stabilizing agents. Furthermore, these compounds also interfered with the transcriptional mechanism of the RET gene in TT cells, subsequently decreasing the endogenous RET protein expression. In present study, we demonstrated the specificity of NSC311152 by using papillary thyroid carcinoma (PTC) cells, the K1, which carry BRAF and PIK3CA mutations but lacks the dominant-activating mutations in this RET proto-oncogene. NSC311152 selectively suppresses cell proliferation by inhibiting the intracellular Raf/MEK/ERK and PI3K/Akt/mTOR signaling pathways in the TT cells. The systemic administration of NSC311152 in a mouse MTC xenograft model significantly inhibited the tumor growth through RET downregulation, whereas NSC31152 only showed a moderate antitumor activity against K1 xenograft tumors. Citation Format: Daekyu Sun, Vishnu Muthuraj Kumarasamy, Tariq Alqahtani. Selective antitumor activity of NSC311152 toward medullary thyroid cancer by downregulating the transcription of RET oncogene [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4783.

Abstract 501: A RET-related microRNA, miR-153-3p, acts as a tumor suppressor in medullary thyroid carcinoma (MTC) via S6K signaling

Abstract Background Medullary thyroid carcinoma (MTC), which comprises 3-5% of all thyroid cancers, originates from a small population of neuroendocrine calcitonin C-cells of thyroid gland. Gain-of-function mutations of the human Rearranged during Transfection (RET) proto-oncogene have been recognized as the key driver of MTC tumorigenesis. RET has been targeted by tyrosine kinase inhibitors (TKIs), however with modest efficacy. Many of the microRNAs (miRNAs) identified in human genome have been associated with cancer and have prompted interest in the establishment of miRNA-based therapeutics. However miRNA studies in MTC has been hampered due to the lack of normal C-cell tissue as a differential expression comparator. Aims We aimed to characterize differentially expressed miRNAs, which play fundamental roles in MTC tumorigenesis and in modulation of TKI responses. Methods RET of human MTC cells were silenced using TKI, Cabozantinib, or siRNA. Small RNA sequencing was performed (AGRF) to identify differentially expressed miRNAs before and after RET inhibition. MiRNA effects on cell proliferation (MTS assay), cell cycle and apoptosis (flow cytometry, Western blot) were investigated with gain-of-function studies. For in vivo study, MTC xenografts were established and miRNAs were intravenously delivered using nanoparticle delivery vehicles invented by biotechnology company - EnGeneIC Ltd. Results miR-153-3p was identified to be under-expressed in MTC following RET inhibition. Restoration of miR-153-3p significantly reduced cell proliferation, induced G2 cell arrest and increased cell apoptosis. Furthermore, combined treatment of miR-153-3p and Cabozantinib caused greater cell proliferation inhibition compared to individual treatment. miR-153-3p targets ribosomal protein S6 kinase B1 (RPS6KB1) and reduced phosphorylation of Bcl-2 associated death promoter (BAD) protein. In vivo delivery of miR-153-3p significantly impeded tumor growth compared to scrambled sequence. Combination of miR-153-3p delivery and oral administration of Cabozantinib resulted in further tumor stabilization effect. Conclusion miR-153-3p reveals a significant tumor suppressive role in MTC via S6K signaling, demonstrating its important therapeutic potential for MTC patients. Citation Format: Lauren Jin Suk Joo, Jocelyn Weiss, Anthony J. Gill, Roderick Clifton-Bligh, Himanshu Brahmbhatt, Jennifer A. MacDiarmid, Matti L. Gild, Bruce G. Robinson, Stan B. Sidhu, Jing Ting Zhao. A RET-related microRNA, miR-153-3p, acts as a tumor suppressor in medullary thyroid carcinoma (MTC) via S6K signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 501.

Effect of parathyroid hormone on apoptosis of human medullary thyroid carcinoma cells*

Abstract Objective The aim of the study was to investigate the effect of parathyroid hormone (PTH) on the apoptosis of human medullary thyroid carcinoma (MTC) cells. Methods In vitro cultured medullary thyroid carcinoma cell lines were treated with parathyroid hormone and parathyroid hormone receptor-monoclonal antibody, and the apoptosis of cells was detected by flow cytometry. Results The cell morphology changed significantly after treatment based on the observation using the inverted phase-contrast microscope. Various concentrations of parathyroid hormone and parathyroid hormone receptor-monoclonal antibody effectively induced apoptosis in a time- and concentration-dependent manner. When the concentration of parathyroid hormone was 2.0 µmol/L and that of parathyroid hormone receptor-monoclonal antibody was 1.0 µmol/L, the apoptotic rate was 13.24% and 20.78%, respectively, representing a statistically significant difference from that of the control cells (P &lt; 0.05). Conclusion PTH plays a role in inducing apoptosis of human MTC cells.

Vandetanib and cabozantinib potentiate mitochondria-targeted agents to suppress medullary thyroid carcinoma cells

ABSTRACTAlthough the FDA-approved receptor tyrosine kinases inhibitors, vandetanib and cabozantinib, are used to treat surgically inoperable progressive medullary thyroid carcinoma (MTC), not all patients are responsive while the disease sometimes progresses after an initial response. To better understand MTC drug resistance at molecular and biochemical levels, we have generated drug-resistant subpopulations of the human MTC cell lines, TT and MZ-CRC-1, via prolonged exposure to vandetanib and cabozantinib. These drug-resistant progenies exhibited substantial cross-resistance to vandetanib and cabozantinib, suggesting that these inhibitors may invoke an overlapping resistance mechanism(s) in MTC cells. Of note, vandetanib and cabozantinib increased mitochondrial membrane potential (Δψm) in drug-naïve as well as drug-resistant cells but only drug-naïve cells exhibited substantially altered oxygen consumption and extracellular acidification rates. Therefore, these inhibitors appear to cause a bioenergetics stress to which drug-resistant MTC cells are more tolerant. Given the ability of vandetanib and cabozantinib to increase Δψm, we hypothesized that these inhibitors can augment growth inhibitory effects of mitochondria-targeted carboxy-proxyl and ubiquinone by increasing their Δψm–dependent uptake/retention in MTC cells. Indeed, our in vitro and mouse xenograft data strongly support this possibility.

Multilayer OMIC Data in Medullary Thyroid Carcinoma Identifies the STAT3 Pathway as a Potential Therapeutic Target in RETM918T Tumors.

Purpose: Medullary thyroid carcinoma (MTC) is a rare disease with few genetic drivers, and the etiology specific to each known susceptibility mutation remains unknown. Exploiting multilayer genomic data, we focused our interest on the role of aberrant DNA methylation in MTC development.Experimental Design: We performed genome-wide DNA methylation profiling assessing more than 27,000 CpGs in the largest MTC series reported to date, comprising 48 molecularly characterized tumors. mRNA and miRNA expression data were available for 33 and 31 tumors, respectively. Two human MTC cell lines and 101 paraffin-embedded MTCs were used for validation.Results: The most distinctive methylome was observed for RETM918T-related tumors. Integration of methylation data with mRNA and miRNA expression data identified genes negatively regulated by promoter methylation. These in silico findings were confirmed in vitro for PLCB2, DKK4, MMP20, and miR-10a, -30a, and -200c. The mutation-specific aberrant methylation of PLCB2, DKK4, and MMP20 was validated in 25 independent MTCs by bisulfite pyrosequencing. The methylome and transcriptome data underscored JAK/Stat pathway involvement in RETM918T MTCs. Immunostaining [immunohistochemistry (IHC)] for the active form of signaling effector STAT3 was performed in a series of 101 MTCs. As expected, positive IHC was associated with RETM918T-bearing tumors (P < 0.02). Pharmacologic inhibition of STAT3 activity increased the sensitivity to vandetanib of the RETM918T-positive MTC cell line, MZ-CRC-1.Conclusions: Multilayer OMIC data analysis uncovered methylation hallmarks in genetically defined MTCs and revealed JAK/Stat signaling effector STAT3 as a potential therapeutic target for the treatment of RETM918T MTCs. Clin Cancer Res; 23(5); 1334-45. ©2016 AACR.

Anti-tumor effects of shikonin derivatives on human medullary thyroid carcinoma cells.

New treatment options are needed for medullary thyroid carcinoma (MTC), a highly metastasizing neuroendocrine tumor that is resistant to standard radiotherapy and chemotherapy. We show that the following shikonin derivatives inhibit cell proliferation and cell viability of the MTC cell line TT: acetylshikonin, β,β-dimethylacrylshikonin, shikonin and a petroleum ether extract of the roots of Onosma paniculata containing several shikonin derivatives. The unsubstituted shikonin derivative was found to be the most effective compound with an IC50 of 1.1 µM. The cell viability of normal human skin fibroblasts, however, was not affected by the tested substances, indicating that shikonin derivatives might be selectively toxic for cancer cells. We further report that migration and invasion of TT cells were inhibited at non-toxic concentrations. Finally, shikonin was tested in vivo using the chick chorioallantoic membrane assay, where it significantly reduced tumor growth by inhibiting cell proliferation and inducing apoptosis. In summary, our results suggest that shikonin derivatives have the potential for the treatment of medullary thyroid carcinomas.

Overexpression of NDRG2 Increases Iodine Uptake and Inhibits Thyroid Carcinoma Cell Growth In Situ and In Vivo.

Medullary thyroid carcinoma (MTC) is an uncommon and highly aggressive tumor of the neuroendocrine system, which derives from the neuroendocrine C cells of the thyroid gland. Except for surgical resection, there are not very many effective systemic treatment options for MTC. N-Myc downstream-regulated gene 2 (NDRG2) had a significantly lower expression in MTC compared with normal thyroid tissue. However, the function of NDRG2 in MTC oncogenesis is largely unknown. In this study, we found that overexpression of NDRG2 inhibited the proliferation of TT cells (human medullary thyroid carcinoma cells) in vitro and suppressed the development of MTC in a nude mouse xenograft model. Further analysis revealed that NDRG2 arrested the cell cycle G0/G1 phase progression and induced TT cell apoptosis. Moreover, NDRG2 overexpression may mediate the antiproliferative effect by reducing cyclin D1 and cyclin E protein levels. We also found aberrant NDRG2-mitigated TT cell migration and invasion in vitro. Sodium/iodide symporter (NIS) mediates active I− transport into the thyroid follicular cells, and radionuclide treatment is a promising therapy for MTC. Our current data revealed that NDRG2 overexpression enhanced NIS level in TT cells and increased their iodine uptake in vitro. Furthermore, 99mTcO4 − radionuclide imaging of the xenograft tumors indicated that NDRG2 could promote NIS-mediated radionuclide transport. In conclusion, the present study suggested that NDRG2 is a critical molecule in the regulation of MTC biological behavior and a potential promoter in radioactive iodine therapy.

Igf-I influences everolimus activity in medullary thyroid carcinoma.

Medullary thyroid carcinoma (MTC) is a rare tumor originating from thyroid parafollicular C cells. It has been previously demonstrated that insulin-like growth factor I (IGF-I) protects MTC from the effects of antiproliferative drugs. Everolimus, an mTOR inhibitor, has shown potent antiproliferative effects in a human MTC cell line, TT, and in two human MTC primary cultures. To verify whether IGF-I may influence the effects of everolimus in a group of human MTC primary cultures. We collected 18 MTCs that were dispersed in primary cultures, treated without or with 10 nM-1 μM everolimus and/or 50 nM IGF-I. Cell viability was evaluated after 48 h, and calcitonin (CT) secretion was assessed after a 6 h incubation. IGF-I receptor downstream signaling protein expression profile was also investigated. Everolimus significantly reduced cell viability in eight MTC [by ~20%; P < 0.01 vs. control; everolimus-responders (E-R) MTCs], while cell viability did not change in 10 MTCs [everolimus-non-responders (E-NR) MTCs]. In E-R MTCs, IGF-I blocked the antiproliferative effects of everolimus that did not affect CT secretion, but blocked the stimulatory effects of IGF-I on this parameter. IGF-I receptor downstream signaling proteins were expressed at higher levels in E-NR MTC as compared to E-R MTCs. IGF-I protects a subset of MTC primary cultures from the antiproliferative effects of everolimus and stimulates CT secretion by an mTOR mediated pathway that, in turn, may represent a therapeutic target in the treatment of aggressive MTCs.

TIM16 inhibition enhances sensitivity to paclitaxel and decreases calcitonin secretion by reducing mitochondrial membrane potential in a human medullary thyroid carcinoma cell line

TIM16 inhibition enhances sensitivity to paclitaxel and decreases calcitonin secretion by reducing mitochondrial membrane potential in a human medullary thyroid carcinoma cell line

Differential expression of cell cycle regulators in CDK5-dependent medullary thyroid carcinoma tumorigenesis.

Medullary thyroid carcinoma (MTC) is a neuroendocrine cancer of thyroid C-cells, for which few treatment options are available. We have recently reported a role for cyclin-dependent kinase 5 (CDK5) in MTC pathogenesis. We have generated a mouse model, in which MTC proliferation is induced upon conditional overexpression of the CDK5 activator, p25, in C-cells, and arrested by interrupting p25 overexpression. Here, we identify genes and proteins that are differentially expressed in proliferating versus arrested benign mouse MTC. We find that downstream target genes of the tumor suppressor, retinoblastoma protein, including genes encoding cell cycle regulators such as CDKs, cyclins and CDK inhibitors, are significantly upregulated in malignant mouse tumors in a CDK5-dependent manner. Reducing CDK5 activity in human MTC cells down-regulated these cell cycle regulators suggesting that CDK5 activity is critical for cell cycle progression and MTC proliferation. Finally, the same set of cell cycle proteins was consistently overexpressed in human sporadic MTC but not in hereditary MTC. Together these findings suggest that aberrant CDK5 activity precedes cell cycle initiation and thus may function as a tumor-promoting factor facilitating cell cycle protein expression in MTC. Targeting aberrant CDK5 or its downstream effectors may be a strategy to halt MTC tumorigenesis.

The expression of the truncated isoform of somatostatin receptor subtype 5 associates with aggressiveness in medullary thyroid carcinoma cells

The truncated somatostatin receptor variant sst5TMD4 associates with increased invasiveness and aggressiveness in breast cancer. We previously found that sst5 activation may counteract sst2 selective agonist effects in a medullary thyroid carcinoma (MTC) cell line, the TT cells, and that sst5TMD4 is overexpressed in poorly differentiated thyroid cancers. The purpose of this study is to evaluate sst5TMD4 expression in a series of human MTC and to explore the functional role of sst5TMD4 in TT cells. We evaluated sst5TMD4 and sst5 expression in 36 MTC samples. Moreover, we investigated the role of sst5TMD4 in TT cells evaluating cell number, DNA synthesis, free cytosolic calcium concentration ([Ca(2+)]i), calcitonin and vascular endothelial growth factor levels, cell morphology, protein expression, and invasion. We found that in MTC the balance between sst5TMD4 and sst5 expression influences disease stage. sst5TMD4 overexpression in TT cells confers a greater growth capacity, blocks sst2 agonist-induced antiproliferative effects, modifies the cell phenotype, decreases E-cadherin and phosphorylated β-catenin levels, increases vimentin, total β-catenin and phosphorylated GSK3B levels (in keeping with the development of epithelial to mesenchymal transition), and confers a greater invasion capacity. This is the first evidence indicating that sst5TMD4 is expressed in human MTC cells, where it associates with more aggressive behavior, suggesting that sst5TMD4 might play a functionally relevant role.

Selective Mitochondrial Uptake of MKT-077 Can Suppress Medullary Thyroid Carcinoma Cell Survival In Vitro and In Vivo.

BackgroundMedullary thyroid carcinoma (MTC) is a neuroendocrine tumor mainly caused by mutations in the rearranged during transfection (RET) proto-oncogene. Not all patients with progressive MTC respond to current therapy inhibiting RET, demanding additional therapeutic strategies. We recently demonstrated that disrupting mitochondrial metabolism using a mitochondria-targeted agent or by depleting a mitochondrial chaperone effectively suppressed human MTC cells in culture and in mouse xenografts by inducing apoptosis and RET downregulation. These observations led us to hypothesize that mitochondria are potential therapeutic targets for MTC. This study further tests this hypothesis using1-ethyl-2-[[3-ethyl-5-(3-methylbenzothiazolin-2-yliden)]-4-oxothiazolidin-2-ylidenemethyl] pyridinium chloride (MKT-077), a water-soluble rhodocyanine dye analogue, which can selectively accumulate in mitochondria.MethodsThe effects of MKT-077 on cell proliferation, survival, expression of RET and tumor protein 53 (TP53), and mitochondrial activity were determined in the human MTC lines in culture and in mouse xenografts.ResultsMKT-077 induced cell cycle arrest in TT and MZ-CRC-1. Intriguingly, MKT-077 also induced RET downregulation and strong cell death responses in TT cells, but not in MZ-CRC-1 cells. This discrepancy was mainly due to the difference between the capacities of these cell lines to retain MKT-077 in mitochondria. The cytotoxicity of MKT-077 in TT cells was mainly attributed to oxidative stress while being independent of TP53. MKT-077 also effectively suppressed tumor growth of TT xenografts.ConclusionMKT-077 can suppress cell survival of certain MTC subtypes by accumulating in mitochondria and interfering with mitochondrial activity although it can also suppress cell proliferation via other mechanisms. These results consistently support the hypothesis that mitochondrial targeting has therapeutic potential for MTC.

Noncoding RNA blockade of autophagy is therapeutic in medullary thyroid cancer.

Micro-RNAs are dysregulated in medullary thyroid carcinoma (MTC) and preliminary studies have shown that miRNAs may enact a therapeutic effect through changes in autophagic flux. Our aim was to study the in vitro effect of miR-9-3p on MTC cell viability, autophagy and to investigate the mRNA autophagy gene profile of sporadic versus hereditary MTC. The therapeutic role of miR-9-3p was investigated in vitro using human MTC cell lines (TT and MZ-CRC-1 cells), cell viability assays, and functional mechanism studies with a focus on cell cycle, apoptosis, and autophagy. Post-miR-9-3p transfection mRNA profiling of cell lines was performed using a customized, quantitative RT-PCR gene array card. This card was also run on clinical tumor samples (sporadic: n = 6; hereditary: n = 6) and correlated with clinical data. Mir-9-3p transfection resulted in reduced in vitro cell viability; an effect mediated through autophagy inhibition. This was accompanied by evidence of G2 arrest in the TT cell line and increased apoptosis in both cell lines. Atg5 was validated as a predicted miR-9-3p mRNA target in TT cells. Post-miR-9-3p transfection array studies showed a significant global decline in autophagy gene expression (most notably in PIK3C3, mTOR, and LAMP-1). Autophagy gene mRNAs were generally overexpressed in sporadic (vs. hereditary MTC) and Beclin-1 overexpression was shown to correlate with residual disease. Autophagy is a tumor cell survival mechanism in MTC that when disabled, is of therapeutic advantage. Beclin-1 expression may be a useful prognostic biomarker of aggressive disease.